Alterations in cellular and organellar phospholipid compositions of HepG2 cells during cell growth

Abstract

The human hepatoblastoma cell line, HepG2, has been used for investigating a wide variety of physiological and pathophysiological processes. However, less information is available about the phospholipid metabolism in HepG2 cells. In the present report, to clarify the relationship between cell growth and phospholipid metabolism in HepG2 cells, we examined the phospholipid class compositions of the cells and their intracellular organelles by using enzymatic fluorometric methods. In HepG2 cells, the ratios of all phospholipid classes, but not the ratio of cholesterol, markedly changed with cell growth. Of note, depending on cell growth, the phosphatidic acid (PA) ratio increased and phosphatidylcholine (PC) ratio decreased in the nuclear membranes, the sphingomyelin (SM) ratio increased in the microsomal membranes, and the phosphatidylethanolamine (PE) ratio increased and the phosphatidylserine (PS) ratio decreased in the mitochondrial membranes. Moreover, the mRNA expression levels of enzymes related to PC, PE, PS, PA, SM and cardiolipin syntheses changed during cell growth. We suggest that the phospholipid class compositions of organellar membranes are tightly regulated by cell growth. These findings provide a basis for future investigations of cancer cell growth and lipid metabolism.

Figure 1

Cell growth of HepG2 cells. Cells were seeded at a density of 5.0 × 10⁴ cells/cm² in six-well plates (Day 0). (a,b) Growth curves of HepG2 cells were determined by the measurement of total cellular DNA (a) and total cellular protein (b). Each point represents the mean value of three biologically independent experiments of duplicate measurements (mean ± S.E., n = 4). (c) Differential interference contrast images of HepG2 cells at Day 3 (early logarithmic phase), Day 6 (late logarithmic phase) and Day 12 (stationary phase). The scale bar represents 100 μm.

Figure 2

Alteration of phospholipid composition in HepG2 cells. HepG2 cells were seeded at a density of 5.0 × 10⁴ cells/cm² in 75-cm² flasks and cultured in DMEM containing 10% FBS at 37 °C for the indicated days. At Day 3 (early logarithmic phase), Day 6 (late logarithmic phase) and Day 12 (stationary phase), cellular lipids were extracted. The contents of PC, PE, PS, PA, PI, PG + CL, SM and cholesterol in HepG2 cells were determined by the enzymatic measurements and protein assay (see Supplementary Table S1). The ratios of PC/TPL (a), PE/TPL (b), PS/TPL (c), PA/TPL (d), PI/TPL (e), (PG + CL)/TPL (f), SM/TPL (g) and cholesterol/TPL (h) at Day 3, Day 6 and Day 12 were evaluated. The total phospholipid (TPL) content (i) was calculated as the sum of PC, PE, PS, PA, PI, PG + CL and SM contents (mean ± S.E., n = 3, *P < 0.05, significantly different among Day 3, Day 6 and Day 12, one-way ANOVA followed by the Bonferroni test). (j) The phospholipid compositions in HepG2 cells at Day 3, Day 6 and Day 12 are shown as pie charts.

Figure 3

mRNA expression changes of phospholipid synthesis-related enzymes in HepG2 cells during cell growth. Relative mRNA expression of CCTA, CCTB, CPT and PEMT (a), that of ECT, EPT, PSD1, PSS1 and PSS2 (b), that of DGKA, DGKG, DGKD, DGKE, DGKQ, DGKK, PLD1 and PLD2 (c), that of CDS1, CDS2, PIS, PGS1 and CLS (d), and that of SPTLC2, SMS1 and SMS2 (e) in HepG2 cells at Day 3 (early logarithmic phase), Day 6 (late logarithmic phase) and Day 12 (stationary phase). The Ct value was normalized to the mean Ct value of HPRT1 and RPLP0. The mRNA expression relative to the mRNA expression level at Day 3 was measured (mean ± S.E., n = 3, *P < 0.05, significantly different among Day 3, Day 6 and Day 12, one-way ANOVA followed by the Bonferroni test).

Figure 4

Isolation of organellar fractions from HepG2 cells. The whole-cell (Whole), purified nuclear (Nuc), microsomal (Mic) and mitochondrial (Mit) fractions (2.0 μg of protein) were separated by 7% or 15% SDS-PAGE and then immunoblotted with anti-NUP98, anti-CNX or anti-COX IV antibody. NUP98, CNX and COX IV are markers of the nucleus, ER and mitochondria, respectively. Three blots were cropped from different PVDF membranes. The full-length blots are presented in Supplementary Fig. S4.

Figure 5

Alteration of nuclear phospholipid composition in HepG2 cells during cell growth. HepG2 cells were seeded at a density of 5.0 × 10⁴ cells/cm² in 150-cm² flasks and cultured in DMEM containing 10% FBS at 37 °C for the indicated days. At Day 3 (early logarithmic phase) and Day 12 (stationary phase), nuclear fractions were isolated from the cells, and nuclear lipids were extracted. The total phospholipid (TPL) content was calculated as the sum of PC, PE, PS, PA, PI, PG + CL and SM contents. The ratios of PC/TPL (a), PE/TPL (b), PS/TPL (c), PA/TPL (d), PI/TPL (e), (PG + CL)/TPL (f), SM/TPL (g) and cholesterol/TPL (h) in the nuclear fractions purified from the cells at Day 3 and Day 12 were determined by the enzymatic measurements (mean ± S.E., n = 3, *P < 0.05, significantly different between Day 3 and Day 12, unpaired two-tailed Student’s t-test). (i) The phospholipid compositions of the purified nuclear fractions from HepG2 cells at Day 3 and Day 12 are shown as pie charts.

Figure 6

Alteration of microsomal phospholipid composition in HepG2 cells during cell growth. HepG2 cells were seeded at a density of 5.0 × 10⁴ cells/cm² in 150-cm² flasks and cultured in DMEM containing 10% FBS at 37 °C for the indicated days. At Day 3 (early logarithmic phase) and Day 12 (stationary phase), microsomal fractions were isolated from the cells, and microsomal lipids were extracted. The total phospholipid (TPL) content was calculated as the sum of PC, PE, PS, PA, PI, PG + CL, and SM contents. The ratios of PC/TPL (a), PE/TPL (b), PS/TPL (c), PA/TPL (d), PI/TPL (e), (PG + CL)/TPL (f), SM/TPL (g) and cholesterol/TPL (h) in the microsomal fractions purified from the cells at Day 3 and Day 12 were determined by the enzymatic measurements (mean ± S.E., n = 3, *P < 0.05, significantly different between Day 3 and Day 12, unpaired two-tailed Student’s t-test). (i) The phospholipid compositions of the purified microsomal fractions from HepG2 cells at Day 3 and Day 12 are shown as pie charts.

Figure 7

Alteration of mitochondrial phospholipid composition in HepG2 cells during cell growth. HepG2 cells were seeded at a cell density of 5.0 × 10⁴ cells/cm² in 150-cm² flasks and cultured in DMEM containing 10% FBS at 37 °C for the indicated days. At Day 3 (early logarithmic phase) and Day 12 (stationary phase), mitochondrial fractions were isolated from the cells and mitochondrial lipids were extracted. The total phospholipid (TPL) content was calculated as the sum of PC, PE, PS, PA, PI, PG + CL, and SM contents. The ratios of PC/TPL (a), PE/TPL (b), PS/TPL (c), PA/TPL (d), PI/TPL (e), (PG + CL)/TPL (f), SM/TPL (g) and cholesterol/TPL (h) in the mitochondrial fractions purified from the cells at Day 3 and Day 12 were determined by the enzymatic measurements (mean ± S.E., n = 3, *P < 0.05, significantly different between Day 3 and Day 12, unpaired two-tailed Student’s t-test). (i) The phospholipid compositions of the purified microsomal fractions from HepG2 cells at Day 3 and Day 12 are shown as pie charts.